The two key factors for the generation of the diverse antigen receptor repertoire in the human adaptive immune system are the products of the recombination activating genes 1 and 2 (RAG1 and RAG2). The identification of their homologs, spRAG1L and spRAG2L respectively, in the genome of the purple sea urchin, Strongylocentrotus purpuratus was unexpected as there is no evidence thus far of an adaptive immune system in invertebrates (including echinoderms), i.e. they lack at least one of its hallmarks, a diversified antigen receptor repertoire. [unreadable] During this fiscal year we established an insect cell expression system that allows us to purify recombinant SpRAG2L protein for biochemical studies. We are in the process of establishing an expression system for SpRAG1L, and a co-expression system for both proteins. We continued characterizing the functional properties of spRAG2L. The C-terminus of this protein is predicted to adopt a plant homeo domain-like (PHD) finger fold, a conserved zinc-binding structure that has been shown to interact with histone tails. We found that the PHD domain of spRAG2L by itself, similar to the respective domain of murine RAG2, is indeed able to bind specifically to histone H3 tails, and it exhibited the same characteristics in the context of the full-length SpRAG2L protein. In contrast to murine RAG2 (recognizing H3K4triMe), however, the respective domain of spRAG2L showed strong preference for peptides with a dimethyl modification of lysine 4 (H3K4diMe). Using purified polyclonal antisera against both, spRAG2L, we also determined that it localizes to the cytoplasm and nucleus when expressed ectopically in 293T cells. This localization pattern in combination with the histone tail recognition motif is consistent with a role of SpRag2L in nuclear processes in the context of chromatin. These findings support our hypothesis that the sea urchin RAG proteins indeed act as a DNA recombinase, and that access to defined regions in the sea urchin genome with native chromatin structure might be regulated at the level of histone modifications. Our studies have major implications for the current model of how adaptive immunity evolved in jawed vertebrates, and will help to illuminate conserved features of how V(D)J recombination is tightly controlled to avoid potentially dangerous modifications of the genome.